Topically Applied Hsp90 Blocker 17AAG Inhibits Autoantibody-Mediated Blister-Inducing Cutaneous Inflammation  Stefan Tukaj, Katja Bieber, Konrad Kleszczyński, Mareike Witte, Rebecca Cames, Kathrin Kalies, Detlef Zillikens, Ralf J. Ludwig, Tobias W. Fischer, Michael Kasperkiewicz  Journal of Investigative Dermatology  Volume 137, Issue 2, Pages 341-349 (February 2017) DOI: 10.1016/j.jid.2016.08.032 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Prophylactic or therapeutic treatment with topical 17AAG reduces clinical and histological disease severity in mice with antibody transfer-induced EBA. Clinical scores, calculated as the percentage of surface area of the shaved back covered by EBA lesions, were lower in mice treated with topical 17AAG (500 nmol) on the shaved back either (a) starting before development of skin lesion (prophylactic setting; daily on days 0–12) or (b) when skin lesions had already developed (therapeutic setting; daily on days 8–15) compared with vehicle. Representative clinical presentations of vehicle- and 17AAG-treated mice at the end of the (c) prophylactic (day 12) and (d) therapeutic treatment (day 15). Semiquantitative histological evaluation of dermal neutrophil infiltration showed lower scores (0, 1, 2, and 3 = no, mild, moderate, and severe infiltration, respectively) in 17AAG-treated mice at the end of the (e) prophylactic and (f) therapeutic treatment. Representative images of hematoxylin and eosin-stained back skin sections of vehicle- and 17AAG-treated mice at the end of the (g) prophylactic and (h) therapeutic treatment. Scale bars = 100 μm. Data are expressed as mean ± standard error of the mean (5–6 mice per group). ∗P < 0.05, ∗∗P < 0.01. 17AAG, 17-allylamino-demethoxygeldanamycin; EBA, epidermolysis bullosa acquisita. Journal of Investigative Dermatology 2017 137, 341-349DOI: (10.1016/j.jid.2016.08.032) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Topical application of 17AAG in a mixed prophylactic-therapeutic setting ameliorates clinical and histological disease severity without affecting autoantibody serum levels in mice with immunization-induced EBA. (a) Clinical scores, calculated as percentage of surface area of the ears or the total body covered by EBA lesions, were lower in mice treated with topical 17AAG (1,000 nmol) on both ears (prophylactic setting; every other day over 10 weeks starting 1 day before immunization) and concurrently on any newly developed skin lesion of the body (therapeutic setting; every other day until week 10) compared with vehicle. (b) Representative clinical presentations of vehicle- and 17AAG-treated mice at the end of the treatment (week 10). (c) Semiquantitative histological evaluation of dermal neutrophil infiltration showed lower scores (0, 1, 2, and 3 = no, mild, moderate, and severe infiltration, respectively) in 17AAG-treated mice at the end of the treatment. (d) Representative images of hematoxylin and eosin-stained ear skin sections of vehicle- and 17AAG-treated mice at the end of the treatment. Scale bars = 100 μm. (e) COL7-specific IgG autoantibody serum levels were similar in vehicle- and 17AAG-treated mice at the end of the treatment, as measured by ELISA. The dashed line represents the background level of the assay. Data are expressed as mean ± standard error of the mean (8–9 mice per group). ∗P < 0.05, ∗∗P < 0.01. 17AAG, 17-allylamino-demethoxygeldanamycin; COL7, type VII collagen; EBA, epidermolysis bullosa acquisita; OD, optical density. Journal of Investigative Dermatology 2017 137, 341-349DOI: (10.1016/j.jid.2016.08.032) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Topical 17AAG inhibits NF-κB activation and affects MMP2, MMP9, MMP12, Flii, and Hsp70 expression in the skin of mice with experimental EBA. In comparison with vehicle controls, 17AAG-treated skin of mice showed reduced levels of (a, b) phosphorylated I-κBα, (c–e) MMP2, (f–h) MMP9, (i–k) MMP12, and (l–n) Flii, as well as (o–q) increased levels of Hsp70 at the end of the antibody transfer- and immunization-induced EBA experiments, as analyzed by fluorescent immunohistochemistry and/or qRT-PCR. Immunofluorescent pictures with corresponding higher magnifications show representative staining of the respective markers (in red) and DAPI nuclear counterstaining (in blue) in skin samples from vehicle- and 17AAG-treated mice at the end of the treatment. Scale bars = 50 μm. Data are expressed as relative mean ± standard error of the mean (5–9 mice per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. 17AAG, 17-allylamino-demethoxygeldanamycin; EBA, epidermolysis bullosa acquisita; MMP, matrix metalloproteinase; qRT-PCR, quantitative real-time reverse transcription-PCR. Journal of Investigative Dermatology 2017 137, 341-349DOI: (10.1016/j.jid.2016.08.032) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Inhibitory effects of topical 17AAG on cellular and molecular pathology are confirmed in a local model of experimental EBA. Short-term topical treatment with 17AAG (500 nmol) applied to both ears (daily for 3 days starting 1 day before local anti-COL7 antibody application, causing locally restricted inflammation of the ear) led to (a) lower degree of dermal neutrophil infiltration based on semiquantitative histological evaluation (0, 1, 2, and 3 = no, mild, moderate, and severe infiltration, respectively), reduced cutaneous levels of (b) phosphorylated I-κBα, (c) MMP2, (d) MMP9, (e) MMP12, and (f) Flii, as well as (g) increased skin expression of Hsp70 at the end of the 3-day treatment period compared with vehicle, as analyzed by fluorescent immunohistochemistry and/or qRT-PCR. Data are expressed as (relative) mean ± standard error of the mean (six mice per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. 17AAG, 17-allylamino-demethoxygeldanamycin; COL7, type VII collagen; EBA, epidermolysis bullosa acquisita; MMP, matrix metalloproteinase; qRT-PCR, quantitative real-time reverse transcription-PCR. Journal of Investigative Dermatology 2017 137, 341-349DOI: (10.1016/j.jid.2016.08.032) Copyright © 2016 The Authors Terms and Conditions